The present invention is in the field of cellular biology, and in particular relates to cell lysis.
Specific lysis of target cells is a feature of a number of normal physiological processes. For example, CD8+ cytotoxic T lymphocytes (CTL) are central to the adaptive immune response against viruses, protozoa, intracellular bacteria, and in the rejection of allogeneic grafts. CTL recognize and kill target cells expressing antigen-derived peptides presented by class I major histocompatibility complex (MHC). In vivo, antigen specific CD8+ T cells also exert effector activity through release of soluble cytokines and chemokines, which either signal other effector cells or have direct antiviral activity. Other cells have lytic activity and participate in the elimination of infected cells or, in some circumstances, self derived cells as in autoimmune diseases. These lytic cells include CD4+ T lymphocytes with lytic effects, Natural Killer (NK) cells, Natural Killer T (NKT) cells and lymphokine activated killer (LAK) cells.
A number of cells that have cytotoxic potential express membrane receptors for the Fc region of an antibody molecule. When antibody is specifically bound to a target cell, receptor-bearing lytic cells bind to the target cell-bound antibody Fc region, and subsequently cause lysis of the target cell. Although the lytic cells are not themselves specific, the specificity of antibody directs them to the specific target cells, in a reaction known as antibody-dependent cell-mediated cytotoxicity (ADCC). Among the cells that can mediate ADCC are natural killer (NK) cell, macrophages, monocytes, neutrophils, and eosinophils. Antibodies can also be used to redirect effector lytic cells toward target cells.
Specific lysis of target cells is also a feature of certain aberrant physiological processes. Self-reactive T lymphocytes and antibodies are involved in the destruction of cells in autoimmune disorders such as insulin-dependent (Type 1) diabetes mellitus and autoimmune hemolytic anemia. Graft-versus-host disease (GVDH) develops when immunocompetent lymphocytes are injected into an allogeneic recipient whose immune system is compromised, e.g., a patient who has had radiation exposure or who has leukemia, immunodeficiency disease, or an autoimmune anemia and who is a recipient of an allogeneic bone marrow transplant. The grafted lymphocytes attack the host, whose immunocompromised state prevents an adequate immune response against the graft.
The cytolytic activity of CD8+ T cells has been commonly determined by the 51Chromium (51 Cr) release assay. The standard 51Cr release assay has a number of disadvantages that include high spontaneous release, influence of 51Cr upon the effector cell population, problems with labeling certain cell types, low sensitivity, and health risks associated with gamma irradiation. Assays such as the modified enzyme linked immuno-assay (ELISPOT), which detects the secretion of cytokines following antigenic stimulation, and the use of tetrameric MHC class I complexes have afforded greater sensitivity in the detection of antigen specific CD8+ T cells.
Non-radioactive alternatives to the standard cytotoxic assays have included detection of released intracellular enzymes, calorimetric assays or detailed preparation of reporter cell lines. In addition, assays that employ the use of flow cytometry by the detection of fluorescent dyes for either lymphocyte-target conjugate formation or cytolytic activity have been described. Flow cytometric cytotoxicity assays generally involve the measurement of a fluorochrome released from, or remaining in, pre-labeled effector cells or targets, simultaneously or exclusively. Ideally, these labels should not change the morphology or function of the labeled cells. However, some of the fluorochromes that have been proposed label target cells poorly, require complex manipulations of light scatter properties to discriminate the viable cell population or have a higher spontaneous release compared to standard 51Cr release. In addition, fluorometric techniques which rely on the incorporation of a nucleic acid stain such as propidium iodide, do not account for active phagocytic cells that can take up dead cells in vitro. As a consequence, none of these proposed assays have gained acceptance, or replaced the 51Cr release assay, which remains in widespread use.
To improve the study of in vitro cytolytic function of lytic agents such as cytolytic cells and antibodies, there is a need for a reliable assay that analyzes subpopulations, eliminates the requirement for potentially hazardous radioactive isotopes, offers increased sensitivity, utilizes an efficient label that is detected easily with low spontaneous release and is reproducible. The present invention addresses this need.
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The present invention provides methods of detecting specific lysis of a cell by a lytic agent. The methods generally involve contacting a labeled target cell with a lytic agent; and detecting fluorescence in the target cell. The target cells are labeled with two fluorescent labels: a fluorescent label that labels the plasma membrane; and a fluorescent label that labels the cytosol. Release of the cytosolic label from the target cell indicates that the target cell has been lysed. The invention further provides methods of detecting the presence in a sample of a cell that specifically lyses a target cell. The invention further provides methods of detecting the presence in a sample of an antibody that specifically lyses a target cell. The methods are useful in a variety of applications.